The responses of methanogenic propionate and butyrate degradation to nano-sized MnO2 exposure were explored. The results showed that supplementation with 50 mg/g volatile suspended solids (VSS) of nano-sized MnO2 significantly enhanced the production rate of CH4 in propionate and butyrate degradation by 25.6% and 21.7%, respectively. The stimulatory effects most likely resulted from enhancements in the microbial metabolic activity based on the observed increases in the extracellular polymeric substance (EPS) secretion and activity of the electron transport system. In contrast, the CH4 yields obtained were irreversibly inhibited by the presence of 400 mg/g VSS of nano-sized MnO2, in which just 62.8% and 6.5%, respectively, of the yield obtained from the control. Further investigations indicated that supplementation by nano-sized MnO2 could cause oxidative stress in microbial cells, resulting in the release of reactive oxygen species (ROS). Compared with that of the control, the amount of intracellular ROS generated in the systems increased by 28.3% (fed with propionate) and 42.5% (fed with butyrate), corresponding to approximately 43.9% and 64.8% losses in cell viability, respectively; thus, ROS generation was suggested to be the main factor responsible for the inhibitory effects of nano-sized MnO2 on methanogenic propionate and butyrate degradation.